Ebselen and diorganylchalcogenides decrease in vitro glutamate uptake by RAT brain slices: Prevention by DTT and GSH

Abstract

The purpose of this study was to investigate the possible involvement of the glutamatergic system in the neurotoxicity of diorganylchalcogenides or organochalcogenides from slices of cerebral cortex in different ages of development: 12- and 60-day-old rats. Glutamate uptake was evaluated in cortical slices of 12 and 60 days old rats. Cortex slices were incubated with three different organochalcogenides with or without reduced glutathione or dithiothreitol. At 100 μM, ebselen, diphenyl diselenide (PhSe)₂ and diphenyl ditelluride (PhTe)₂ in vitro inhibited the [³H]glutamate uptake in both age. Both 60-day-old rats and for 12-day-old rats, GSH and DTT prevented the (PhTe)₂-induced inhibition of glutamate uptake but did not protect the inhibition caused by ebselen and (PhSe)₂. These findings suggest that the neurotoxicity of organochalcogenides could be related to their effects on brain glutamate uptake, conceivably involving a redox modulation of reactive amino acids from the glutamate transporter proteins.

Introduction

Evidence has been provided in the last two decades indicating that organochalcogenides, as the organoselenium and organotellurium compounds are promising pharmacological agents and to possess very interesting biological activities. Despite the well established neurotoxic activities related to organochalcogens, several reports have been published on glutathione peroxidase (GPx)-mimetic activity of organochalcogenides compounds, which, like the native enzyme, rely on the redox cycling of selenium or tellurium moiety of the compounds (Parnham and Sies, 2000, Klotz and Sies, 2003, Klotz et al., 2003, Nogueira et al., 2004). Thus, a variety of organochalcogenides (including diphenyl ditelluride and diphenyl diselenide) can decompose H₂O₂ or a variety of hydroperoxides from lipids to H₂O or their equivalent alcohols using GSH or other synthetic reduced thiols as electron donors (Nogueira et al., 2004). In fact, ebselen (a mimetic of GPx) has been demonstrated to exert a protective role against brain ischemia and stroke (Dawson et al., 1995, Yamaguchi et al., 1998, Daiber et al., 2000) and, in experimental models, against glutamate excitotoxicity (Porciuncula et al., 2001, Porciuncula et al., 2003, Rossato et al., 2002a, Rossato et al., 2002b) and after in vivo and in vitro exposure to methylmercury (Farina et al., 2003a, Farina et al., 2003b, Moretto et al., 2004). Ebselen may be neuroprotective in part due to its actions as a modulator of the NMDA receptor redox modulatory site. Its putative mode of action as a neuroprotectant is via cyclical reduction and oxidation reactions, in a manner like to glutathione peroxidase (Herin et al., 2001. In addition, Ebselen has been shown to produce in vitro protective effects on the excitotoxic injury in retina cells and on the [3H]glutamate uptake in human platelets (Borges et al., 2004, Centurião et al., 2005).

In this context, we have recently demonstrated that diphenyl diselenide (PhSe)₂, a simple organochalcogenide, has neuroprotective and anti-inflammatory activity (Ghisleni et al., 2003, Nogueira et al., 2003a, Nogueira et al., 2003b, Nogueira et al., 2004, Zasso et al., 2005). The neuroprotective effect of diphenyl diselenide seems to be related at least in part to its capacity to diminish the expression of iNOS induced by oxygen and glucose deprivation in brain slices (Ghisleni et al., 2003). Furthermore, diphenyl diselenide can also enhance the memory of rodents, which may be related to neuroprotective actions of this compound (Rosa et al., 2003). The mechanisms that underlie the anti-inflammatory and antinoceptive action of diphenyl diselenide are not well characterized but may involve several targets, including inhibition of iNOS, reduction of hydroperoxide tone and the synthesis of inflammatory intermediaries derived from arachidonic acid (Nogueira et al., 2004). Indeed, their mechanisms of action are not exactly equivalent (Moretto et al., 2003). Recently, data from our laboratory have shown that the selenium compounds ebselen and diselenide present protective actions toward the alterations of the phosphorylating system associated with the intermediate filaments proteins induced by methylmercury in slices of the cerebral cortex of rats (Moretto et al., 2005a).

Inorganic and organic tellurium, particularly the diphenyl ditelluride, are extremely neurotoxic compounds (Jackson et al., 1989, Maciel et al., 2000, Nogueira et al., 2001, Stangherlin et al., 2005), while diphenyl diselenide can be neurotoxic to rodents only after exposure to high doses (Maciel et al., 2000, Jacques-Silva et al., 2001, Nogueira et al., 2003a). Nevertheless, the analogous compound of diphenyl diselenide, diphenyl ditelluride, caused a stimulation of phosphorylation that was blocked by diphenyl diselenide and ebselen (Moretto et al., 2005b).

Glutamate, the main excitatory neurotransmitter in the mammalian brain, plays important roles in several physiological processes (Ozawa et al., 1998) such as memory and learning (Izquierdo and Medina, 1997), development and ageing (Segovia et al., 2001) and adaptation to the environment (Danbolt, 2001, Warren, 2002). Glutamate uptake is the process responsible for the maintenance of extracellular glutamate concentrations below neurotoxic levels. The subtypes of glutamate transporters are expressed regionally in distinct patterns, with glial and neuronal transporter expression appearing in coordinated manner during CNS development (Ullensvang et al., 1997, Furuta et al., 1997). Glutamate toxicity has been related to neuronal death in ischemia, hypoxia, hypoglycemia and trauma (Choi, 1988, Ikonomidou et al., 1989) and with many chronic neurodegenerative disorders of the CNS, including Huntington’s and Alzheimer’s, and status epilepticus (Brewer, 2000, Maragakis and Rothstein, 2004, Sheng et al., 2005).

The aims of the present investigation were: (1) to compare the effects of ebselen, diphenyl diselenide and diphenyl ditelluride on glutamate uptake by brain cortical slices with the purpose to evaluate whether the differential neurotoxicity of these compounds could be attributed at least in part to their action on the glutamatergic system; (2) to observe the effect of reduced glutathione (GSH) or dithiothreitol (DTT) on the organochalcogenides action on glutamate uptake, and (3) to determine whether ontogenetic development modifies the sensitivity of glutamate uptake to these agents; this is important because developing brain can be more sensitive to external insults, particularly to tellurium (Perez-D’Gregorio and Miller, 1988, Stangherlin et al., 2005).